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INSERM U-349, Hôpital Lariboisiére (S.R., F.P., A.L., C.M., A.J., M.-C.d.V.), Paris, France; and Nuffield Hospital (J.Q.), Oxford, United Kingdom
Address all correspondence and requests for reprints to: M. C. de Vernejoul, M.D., INSERM U-349, Hôpital Lariboisière, 6 rue Guy Patin, 75475 Paris Cedex 10, France.
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Abstract |
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In conclusion, our results suggest that PGE2 has an inhibitory effect on human OC differentiation from CBMs, possibly by reducing precursor proliferation in these cultures. We also hypothesize that PGE2 may reduce OC differentiation by increasing the proportion of precursor cells that differentiate into macrophages. In addition, this may be the result of inhibition of the c-fos expression in CBMs.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostaglandins (PGs) are lipid mediators that play an important role in bone metabolism. They are potent stimulators of bone resorption in bone organ cultures (1, 2) and when administrated in vivo in mice (3). In addition, a congenital condition associated with PGE hyperproduction has been described in children, characterized by hypercalciuria, nephrocalcinosis, and osteopenia, suggesting a role for PGs in the observed bone loss (4, 5). Contrary to these observations, PGs exert an inhibitory effect on isolated OCs (6, 7). The results of studies on murine osteoclast differentiation, using bone marrow cultures or cocultures of hematopoietic and stromal cells, indicate that PGs play an important role in OC differentiation, although complex; in fact, PGs appear to be either stimulator (8, 9, 10) or inhibitor (11), depending on the culture system, particularly the nature of the stromal cell supporting the OC differentiation (12). These dual effects of PGs may reflect a cytokine-mediated response through intermediate cells such as osteoblasts (13). In human bone marrow cultures, opposite effects of PGs on OC differentiation have also been reported, either stimulatory (14) or inhibitory (15), probably depending on the culture system.
The direct effect of PGE2 on human OC differentiation
using hematopoietic osteoclast precursors cultured alone has never been
evaluated. We have investigated the effect of PGE2 on human
OC differentiation using human cord blood monocytes as a source of
hematopoietic OC precursors. We have previously shown that CBMs, when
cultured for 3 weeks in the presence of 1,25-dihydroxyvitamin
D3 [1,25-(OH)2D3], form
multinucleated cells (MNCs), a significant population of which express
the OC phenotype as they express calcitonin receptor (CTR), vitronectin
receptor (VNR; 
vß3), and
tartrate-resistant acid phosphatase (TRAP) (16). In addition, they may
undergo further differentiation into bone-resorbing cells under the
appropriate conditions. Indeed, we showed that CBMs are capable of
terminal OC differentiation when cultured either in the presence of
culture medium conditioned by giant cell tumor of bone (17) or in a
bone microenvironment by adding CBMs to organ cultures of explanted
bone (18). Some of the cultured CBMs expressed macrophage markers, such
as CD11c and DR antigen (16). CBMs contain precursors cells that can
differentiate in either OC or macrophage.
Our findings concerning the effects of PGE2 on CBMs indicate that PGE2 have a direct inhibitory effect on human OC differentiation and that they enhance the commitment of progenitor cells in the macrophage differentiation at the expense of OC differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBM proliferation
Cord blood monocytes were cultured as described in three
experiments in the presence of PGE2 where appropriate. CBMs
were assessed for proliferation after 7 days of culture by use of a
[3H]thymidine incorporation assay. One microcurie of
[3H]thymidine (SA, 25 Ci/mmol) in 25 µl PBS was added
to each well for 8 h. Cells were then washed and fixed, and
autoradiography was performed. The number of cells with
[3H]thymidine-positive nuclei was counted. Results from
triplicate wells were expressed as a percentage of the labeled cells
(mean ± SEM).
Immunohistochemistry
Immunohistochemistry was performed using an indirect
immunoperoxidase technique to stain cultured cells in six experiments
after a 3-week culture period in the presence of PGE2 where
appropriate. In three other experiments, PGE2
(10-7 M) was added either during a 3-week
culture period or only during the third week of the culture. To detect
membrane antigens expressed by cells of the monocyte-macrophage
lineage, we used antibodies against osteoclast antigen, VNR (monoclonal
antibody against ß3-chain, Pierce, Paris, France) and
macrophage antigens, CD11b and CD14 (monoclonal antibodies anti-CD11b
and anti-CD14, Sera-Lab, Sussex, UK). Results were expressed as a
percentage of the labeled cells (mean ± SEM).
Reverse transcription-PCR (RT-PCR) and analysis of PCR products
To study c-fos, ß3-chain, and CTR
messenger RNA (mRNA) expression, total RNA from seven cord
blood-cultured cells were extracted by RNAzol (Bioprobe System,
Montreuil-sous-Bois, France), a method derived from the extraction
procedure of Chomczynski and Sacchi (19). c-fos and
ß3-chain complementary DNA (cDNA) were synthesized from
1.5 µg total RNA in a 20-µl volume reaction with 200 U Moloney
murine leukemia virus reverse transcriptase (Life Technologies,
Cergy-Pontoise, France) in the presence of 1 mM of each
deoxy-NTP and 50 pmol of a 3'-oligo(deoxythymidine) primer for 60 min
at 37 C. The reaction was then divided in half, and each part was
diluted to 50 µl with the same buffer containing 25 pmol of each
specific set of primers for glyceraldehyde-3-phosphate dehydrogenase
(GADPH; internal control), c-fos or ß3-chain
(Genosys, Cambridge, UK; Table 1
), and 0.75 U
Taq DNA Polymerase (Life Technologies). Amplification was
performed during 25 cycles as follows: 95 C for 30 sec, 55 C for 30
sec, 72 C for 30 sec, and 72 C for 5 min at the end of the 25 cycles.
Amplified products were then analyzed by electrophoresis in 2% agarose
gel, visualized by ethidium bromide, and transferred to nylon membrane
(Southern blot; GeneScreen, DuPont-New England Nuclear Products,
Boston, MA). Each amplified cDNA was hybridized with an appropriate
specific 50-mer oligonucleotide antisense probe (Genosys), labeled with
[32P]deoxy-CTP using an oligonucleotide 3'-end-labeling
system (DuPont-New England Nuclear Products), and purified with Nensorb
cartridges (DuPont-New England Nuclear Products). The membrane was
hybridized at -25 C in the presence of 6 x SSC (standard saline
citrate), 2 x Denhardt’s solution (0.4% Ficoll, 0.4%
polyvinylpyrollidone, 0.4% B5A), 0.1% SDS, then washed at -20 C in
2 x SSC-0.1% SDS. Autoradiography was performed at -80 C with
intensifying screen.
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). Amplification and hybridization of
CTR-amplified cDNA with a CTR-specific cDNA probe radiolabeled with
[32P]dCTP using a random priming method were performed as
previously described (20). Analysis of the amplified products (ß3, c-fos, and h-CTR) was related to the internal control GAPDH, and semiquantification of the amplified products was performed using a densitometer (Transidyne General Corp., Ann Arbor, MI). Signals were normalized using the values obtained for the corresponding GAPDH amplifications. Results were expressed as the ratios of c-fos/GADPH, ß3/GADPH, and CTR/GADPH.
Statistical analysis
Each series of experiments (multinucleation and
immunohistochemistry) was repeated at least six times, except for the
proliferation assay, which was performed in three experiments. Results
are expressed as the mean ± SEM, and significance was
determined using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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).
Effects of PGE2 on CBM phenotype
The expression of macrophage markers (CD11b and CD14) and an OC
marker (ß3-chain) by cultured CBMs was investigated
by immunohistochemistry; results are expressed as the mean proportion
of positive cells ± SEM in six experiments. The
proportion of ß3-positive cells in controls was
33.58 ± 2.78%; the proportion of ß3-positive cells
was significantly decreased in cultures incubated with
10-8, 10-7, and 10-6
M PGE2 [respectively, 24.48 ± 1.51%
(P < 0.05), 13.68 ± 2.6% (P <
0.001), and 12.27 ± 2.27% (P < 0.001)]
compared to that in controls (Fig. 2A). The proportion
of CD14-positive cells in control cultures was 32.86 ± 1.1%; the
proportion of CD14-positive cells was significantly increased in
cultures incubated with 10-8, 10-7, and
10-6 M PGE2 [respectively,
41.1 ± 3.33% (P < 0.05), 52.95 ± 3.36%
(P < 0.001), and 55.37 ± 2.27%
(P < 0.001)] compared to that in controls (Fig. 2B).
The proportion of CD11b-positive cells in controls was 14.22 ±
2.35%; the proportion of CD11b-positive cells was significantly
increased in cultures incubated with 10-8,
10-7, and 10-6 M PGE2
[respectively, 21.7 ± 2.49% (P < 0.05),
27.96 ± 3.91% (P < 0.05), and 38.11 ±
3.93% (P < 0.001)] compared to that in controls in a
dose-dependent manner (Fig. 2C).
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). Total RNAs were extracted from CBMs after a 3-week
culture period in four RT-PCR experiments (Fig. 4A) and
from CBMs cultured for 1, 2, and 3 weeks in two RT-PCR experiments
(Fig. 4B). Results were expressed as the ratio of
ß3/GADPH. PGE2 strongly reduced the
expression of ß3 mRNA from the first week. After a 3-week
culture period, the calculated ratio of ß3/GAPDH PCR
product revealed a decrease of approximately 10-fold for the
PGE2-treated CBMs compared to the untreated cells
(10.32 ± 1.48).
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), both CTR RNA
isoforms, h-CTR1 and h-CTR2, were detected in CBM control cultures,
whereas in the presence of 10-7 M
PGE2, only the h-CTR1 isoform was detected in CBMs. In the
second experiment, hCTR1 was the only isoform detected in both control
and PGE2-treated CBMs. In these two experiments, the amount
of PCR product, normalized using GAPDH amplification, was lower in the
PGE2-treated CBMs compared to the untreated cells, and the
calculated ratio of h-CTR/GAPDH PCR product revealed a decrease of
approximately 6-fold in the PGE2-treated CBMs compared to
the untreated cells (6.45 ± 1).
Effects of PGE2 on c-fos RNA expression
c-fos mRNA expression was evaluated by RT-PCR in CBMs
cultured in the presence or absence of 10-7
M PGE2 after a 3-week culture period (Fig. 3C).
Total RNAs were extracted from CBMs after a 3-week culture period in
three RT-PCR experiments (Fig. 4C), and from CBMs cultured for 1 and 3
weeks in two RT-PCR experiments (Fig. 4D). Results were expressed as
the ratio of c-fos/GADPH. c-fos mRNA expression
was markedly reduced when CBMs were cultured in the presence of
PGE2 compared to that in the control cultures. After a
3-week culture period, the calculated ratio of c-fos/GAPDH
PCR product revealed a decrease of approximately 8-fold in the
PGE2-treated CBMs compared to that in the untreated cells
(7.89 ± 2.3).
Effects of PGE2 on CBM multinucleation, CBM
phenotype, and c-fos expression when added at the third week of
culture
In three experiments, PGE2 was added either for
the 3-week culture period or only during the third week of culture,
when cells were not proliferating (16). This late addition of
PGE2 (10-7 M) did not modify the
expression of CD11b and ß3-chain evaluated by
immunohistochemistry or c-fos mRNA expression evaluated by
RT-PCR compared to the control values. The number of MNCs and the
fusion index, corresponding to the percentage of the total number of
nuclei that were within MNCs, were also unchanged (Fig. 5, A–E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results identify some of the effects of PGE2 on human OC precursor differentiation in an in vitro system using hematopoietic precursor cells in the absence of stromal cells. CBMs in long term culture in the presence of 1,25-(OH)2D3 express characteristics of OCs, including VNR, TRAP, and functional CTR (16, 21), although they need a further stimulus to undergo terminal OC differentiation. Indeed, we have previously shown that CBMs form bone-resorbing cells when cultured in the presence of culture medium conditioned by giant cell tumor of bone or in a bone microenvironment (17, 18). We found that PGE2 added at the beginning of CBM cultures had an inhibitory effect on OC differentiation, as shown by reduced number of cells expressing OC markers (ß3-chain and CTR), as well as reduced multinucleation and proliferation. In addition, we observed that the number of cells expressing CD11b and CD14, macrophage markers that are not expressed by human OC (22), was increased in the presence of PGE2. Such an increased expression of macrophage markers in the presence of PGE2 has been previously reported in mouse bone marrow-derived macrophage cultures (23).
CTR is characteristic of OCs in bone. CTR is also present in postmitotic OC precursors before their terminal differentiation into bone-resorbing cells. CTR is expressed in both late OC precursors and mature OCs (24). In this study, OC differentiation was evaluated by examining the expression of CTR, which is, therefore, a reliable marker for identifying OC differentiation in vitro.
Two isoforms of h-CTR have been identified that are identical except for the presence (h-CTR1) or absence (h-CTR2) of a 16-amino acid insert in the first intracellular loop. These two CTR isoforms are generated by alternative splicing and have different signaling properties; both signal via the adenyl cyclase pathway, but h-CTR2 is also capable of stimulating the phosphoinositide-specific phospholipase C pathway (25). A third h-CTR isoform has recently been identified (26). CTR has been identified in tissue such as kidney, brain, and lung; however, in bone tissue, CTR expression is found only in OCs. The two isoforms, h-CTR1 and h-CTR2, are expressed by OCs from human giant cell tumor of bone (27). We have shown here that mRNA of both CTR isoforms, h-CTR1 and h-CTR2, or only h-CTR1 were expressed in CBMs after a 3-week culture period. In the presence of PGE2, expression of CTR was diminished, and only the h-CTR1 isoform was detected. This differential expression suggested that PGE2 had a direct or indirect effect on the transcriptional regulation of CTR in CBMs, although the consequence of this for OC differentiation is unclear.
The vitronectin receptor (vß3) is strongly
expressed in OCs; however, it is also expressed by many other cells,
including macrophages, although in a low proportion (22). Our
observation of a reduction of cells expressing ß3-chain,
therefore, is consistent with the reduced OC differentiation.
The inhibitory effect of PGE2 on OC differentiation in our CBM culture system is an early event; the reduction of the RNA ß3-chain and CTR expression was evident from the first week of CBM culture. As evidence exists that OC progenitor proliferation precedes and is essential for OC differentiation (28), we, therefore, investigated the effect of PGE2 on CBM proliferation, evaluated on day 7 of the culture period, when maximum CBM proliferation occurs in this system (16). We found an inhibitory effect of PGE2 on CBM proliferation. When PGE2 was present only during the third week of the culture period, no effect was observed on the analyzed markers (multinucleation, fusion index, ß3-chain and CD11b expression, and c-fos mRNA expression). These results indicate that the inhibitory effects of PGE2 on OC differentiation may be due in part to inhibition of OC precursor proliferation.
Fos protein, a product of the c-fos protooncogene, is a component of the AP-1 transcription factor. In an avian bone marrow culture system, mononuclear OC precursors and multinucleated OCs constitutively expressed mRNA c-fos, and c-fos DNA transfection in OC precursor cells stimulated TRAP activity and bone resorption by these cells (29). Our results are consistent with the important role that c-fos plays in OC activity and differentiation. Indeed, we have shown that in addition to its inhibitory effect on OC differentiation, PGE2 decreased mRNA c-fos expression in CBM cultures, and that this inhibition was present from the first week of the culture. The reduced c-fos expression in CBMs observed in the presence of PGE2 suggests that one of the mechanisms by which PGE2 may inhibit OC differentiation is via an effect on c-fos expression in CBMs. There is evidence that leukotriene B4, which belongs to the eicosanoid family, increases the expression of c-fos in human monocytes via an increase in c-fos transcription (30). Our results are consistent with a modulation of c-fos expression by PGE2, but whether PGE2 inhibits OC differentiation in CBMs by decreasing c-fos expression or whether the reduced c-fos expression is the result of a reduction in OC differentiation caused by PGE2 acting via some other mechanism remains to be elucidated. In accordance with the hypothesis that a reduced c-fos expression may inhibit OC differentiation are the results of Grigoriadis et al. (31). These researchers showed that mice lacking the protooncogene c-fos develop a type of osteopetrosis and that OC differentiation is blocked in these mice; mature OCs were absent in mutant mice, although early OC progenitors were identified (31). In addition, Udagawa et al. (32) have shown in a murine coculture system that c-fos antisense DNA inhibited OC formation when added early. Corroborating these results, we found that the addition of PGE2 during the last week of culture did not induce any change in the phenotype of CBMs or in the expression of c-fos.
In CBMs cultured for 3 weeks, we observed that the number of cells expressing macrophage markers was increased and the number of cells expressing OC markers was decreased in the presence of PGE2. PGE2 may, therefore, cause an increased commitment of progenitor cells to macrophage differentiation while inhibiting their differentiation into OCs. These data further emphasize the potential role of the reduced c-fos expression in the inhibitory effect of PGE2 on OC differentiation in our system; in mice lacking the protooncogene c-fos, in addition to the absence of OC formation, the number of bone marrow macrophages was increased, suggesting that the lack of Fos resulted in a lineage shift between OCs and macrophages (31).
In summary, we evaluated the effects of PGE2 on OC differentiation from human OC precursors in the absence of a stromal cell population. We demonstrated that PGE2 reduced the OC differentiation in this model. However, additional studies are needed to elucidate the mechanism underlying the inhibition of OC formation by PGE2.
Received August 26, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(cachectin) stimulates
bone resorption in mouse calvaria via a prostaglandin-mediated
mechanism. Endocrinology 120:2029–2036[Abstract]
on osteoclast formation in mouse bone
marrow cultures. J Bone Miner Res 6:157- 164[Medline]
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)
or presence of PGE2 (10-7 M; 
).
A, CBMs were cultured for 3 weeks in four experiments (no. 1–4). B,
CBMs were cultured for 1, 2, or 3 weeks in two experiments (no. 1 and
2). C, CBMs were cultured for 3 weeks in three experiments (no. 1–3).
D, CBMs were cultured for 1 or 3 weeks in two experiments (no. 1 and
2).
), for only the third week of the culture
(